Anion Exchange ColumnAnion Exchange Column

By: Eilish McConville By: Eilish McConville

Justin LowJustin Low

Presentation OverviewPresentation Overview

• Theory and BackgroundTheory and Background• Column SizingColumn Sizing• TimingTiming• Engineering DesignEngineering Design• CostsCosts• RecommendationsRecommendations

Theory and BackgroundTheory and Background

• Chromatography separation method Chromatography separation method differentiating species based on the differentiating species based on the ionic chargeionic charge

• Four Stages:Four Stages:• Initial BindingInitial Binding• WashWash• EluteElute• RebufferRebuffer

• Elution ProfileElution Profile

Column SizingColumn Sizing

• Calculated Protein Loading vs. VolumeCalculated Protein Loading vs. Volume• Used Empirical data from Bio-RadUsed Empirical data from Bio-Rad• Model:Model:

Protein Load (mg) = 15.007*Volume(mL)-0.125Protein Load (mg) = 15.007*Volume(mL)-0.125

0

20

40

60

80

100

120

140

160

180

200

0 2 4 6 8 10 12 14

Volume (mL)

Pro

tein

Lo

adin

g (

mg

)

Column SizingColumn Sizing

• Pick Suitable ColumnPick Suitable Column• Consider Size, efficiency and costConsider Size, efficiency and cost• GE Chromaflow column selectedGE Chromaflow column selected• Volume = 942.5 LVolume = 942.5 L

• Calculate Total Number of Calculate Total Number of Column Runs for single columnColumn Runs for single column• Total amount of protein/batch = Total amount of protein/batch =

202.80kg202.80kg• Can separate 7.071kg/column runCan separate 7.071kg/column run• Need to run column 28.67 timesNeed to run column 28.67 times

TimingTiming

• Need to consider two time frames:Need to consider two time frames:• Batch Process time for columnBatch Process time for column• Continuous process time for entire insulin Continuous process time for entire insulin

productionproduction

• Process requires multiple columnsProcess requires multiple columns• Chose a flow rate that allowed for four columnsChose a flow rate that allowed for four columns• 93.82 L/min (72% of maximum)93.82 L/min (72% of maximum)• Determined the step times based on flow rateDetermined the step times based on flow rate

Number of ColumnsNumber of Columns

• Four columns is idealFour columns is ideal• Operation can be offset to allow for Operation can be offset to allow for

continuous productcontinuous product

StepStep Volume (L)Volume (L) Time (min)Time (min)

Insulin Ester Insulin Ester solutionsolution

36.8436.84 0.390.39

WashWash 4712.394712.39 50.2350.23

ElutionElution 4712.394712.39 50.2350.23

RebufferRebuffer 4712.394712.39 50.2350.23

TotalTotal 14174.0114174.01 151.08151.08

Engineering DesignEngineering Design

Design ConsiderationsDesign Considerations

• Insulin Ester Isoelectric Point = 5.3Insulin Ester Isoelectric Point = 5.3• Bead Choice:Bead Choice:

• DEAE-Sephadex A-25DEAE-Sephadex A-25• Medium to coarse beads (170-260 Medium to coarse beads (170-260 µm)µm)

• Initial buffer pHInitial buffer pH• Flow rate chosen for four columnsFlow rate chosen for four columns• 50% Protein Saturation50% Protein Saturation

CostCost• GE quoted price = $150,577 (CAN)/columnGE quoted price = $150,577 (CAN)/column• Total Capital Cost ~ $900 000 (CAN)Total Capital Cost ~ $900 000 (CAN)• Amortized Cost (20 years, 10%) ~ $100 Amortized Cost (20 years, 10%) ~ $100

000/year000/year• Operating CostsOperating Costs

• Gel Matrix refills (~$2000/kg or $2.6M/column)Gel Matrix refills (~$2000/kg or $2.6M/column)• Utility costsUtility costs• Wash and Eluent Material CostsWash and Eluent Material Costs• MaintenanceMaintenance

• Other Capital Costs not consideredOther Capital Costs not considered• PumpsPumps• Holding TanksHolding Tanks• Control SystemControl System

ManufacturersManufacturers• GEGE• Bio-Rad Bio-Rad

LaboratoriesLaboratories• Dionex Corp.Dionex Corp.• Shodex Inc.Shodex Inc.• Thermo LabsThermo Labs• Sartorius AGSartorius AG• Sigma AldrichSigma Aldrich• Varian Inc.Varian Inc.• UpFront UpFront

ChromatographyChromatography

RecommendationsRecommendations

• Experimentation with the unit to Experimentation with the unit to determine:determine:• Optimal buffer pHOptimal buffer pH• Protein loadingProtein loading

• Talk with suppliers:Talk with suppliers:• General Electric CompanyGeneral Electric Company• UpFront ChromatographyUpFront Chromatography

QuestionsQuestions